Throughout this application, various publications are referred to. These publications, and references included therein, are incorporated herein in their entirety.
Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the T-cell surface receptors, CD28 and CTLA-4 (CD152). Engagement of CD28 with B7-1 (CD80) and B7-2 (CD86) ligands expressed on antigen-presenting cells provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response [reviewed by Oosterwegel et al., Curr. Opin. Immunol. 11:294-300 (1999); Lenschow et al., Annu. Rev. Immunol. 14:233-258 (1996); Greenfield et al., Crit. Rev. Immunol. 18:389-418 (1998)]. Altering these interactions has profound effects on immune responses in experimental disease models. CTLA-4- and CD28-associated signaling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumor immunity [reviewed by Oosterwegel (1999) ibid.; Ikemizu et al., Immunity 12:51-60 (2000)]. Enhanced anti-tumor immune responses result from transfecting B7-1 into murine tumors or from using anti-CTLA-4 antibodies to block CTLA-4 interactions with B7-1 and B7-2. Conversely, inhibition of B7/CD28 interactions results in general immunosuppression, reduced autoantibody production, and enhanced skin and cardiac allograft survival. There is, therefore, considerable interest in manipulating human B7 interactions, and such approaches have already shown promise [Guinan et al., N. Engl. J. Med. 340:1704-1714 (1999); reviewed by Ikemizu (2000) ibid.].
B7-1 and B7-2 are glycoproteins, each consisting of single V-like and C-like immunoglobulin superfamily (IgSF) domains. Their ligands, CD28 and CTLA-4, are also structurally related and expressed at the cell surface as disulfide-linked homodimers of single V-like IgSF domains. A third CD28-like molecule, ICOS, interacts with another B7-related molecule, but the analysis of transgenic mice indicates that B7-1 and B7-2 are the only functional ligands of CD28 and CTLA-4. The affinities of these interactions differ substantially: human CTLA-4 binds B7-1 with a solution Kd of 0.2-0.4 μM, whereas the affinity of CD28 for B7-2 is 40- to 100-fold lower (B7-1/CD28 and B7-2/CTLA-4 interactions each have intermediate affinities [Kd=4 μM]) [reviewed by Ikemizu (2000) ibid.].
Expression of B7-1, B7-2, CD28 and CTLA-4 is tightly regulated: whereas CD28 is constitutively expressed on resting human T cells and B7-2 is rapidly induced on antigen-presenting cells early in immune responses, the expression of both B7-1 and CTLA-4 is considerably delayed [reviewed by Lenschow (1996) ibid.]. Interactions of the B7. molecules with CD28 generate costimulatory signals amplifying T cell receptor (TCR) signaling and preventing anergy, whereas interactions with CTLA-4 induce powerful inhibitory signals in T cells. CD28-dependent costimulation is poorly understood, but recent work implicates the bulk recruitment of cell surface molecules and kinase-rich rafts to the site of TCR engagement, favoring receptor phosphorylation and signaling. Conversely, CTLA-4 inhibits signal transduction by inducing the dephosphorylation of TCR and RAS signaling pathway components and by interfering with distal events in the CD28 signaling pathway [reviewed by Ikemizu (2000) ibid.].
While the opposing effects of CD28 and CTLA-4 are clear-cut, distinct functions for B7-1 and B7-2 have yet to be defined. A role of Th0, Th1, or Th2 differentiation has been proposed [Freeman et al., Immunity 2:523-532 (1995); Kuchroo et al., Cell 80:707-718 (1995)] but other work suggests that B7-1 and B7-2 determine the magnitude of costimulatory signals rather than the outcome of Th subset differentiation. Moreover, gene disruption studies reveal considerable overlap in the costimulatory functions of B7-1 and B7-2. It has been suggested that rather than having distinct CD28-dependent costimulatory roles, the key functional differences between B7-1 and B7-2 concern strength and/or mode of their binding to CD28 and CTLA-4 [reviewed by Ikemizu (2000) ibid.]. In addition, the differential timing of the expression of B7-2 and B7-1, as well as of CD28 and CTLA-4, already referred to above, is likely to be critical in the early events during a cellular immune response [reviewed by Lenschow (1996) ibid.].
Full T cell activation requires, as said, both an antigen-specific and a second, antigen-independent costimulatory signal. A unique group of antigens is comprised of a family of pyrogenic exotoxins, also known as superantigen toxins, produced by Staphylococcus aureus and Streptococcus pyogenes. The exotoxins comprised of the S. aureus enterotoxins (SEs) cause the majority of human food poisoning cases manifested by vomiting and diarrhea after ingestion [Schlievert, J. Infect. Dis. 167:997 (1993)]. S. aureus is found widespread in nature, often in association with humans. Among the major serological types within the family of SEs (including but not limited to SEA to SEE and SEG), SEB is prominent [Marrack and Kappler, Science 248:705 (1990)]. SEB has also been recognized as a leading cause of human cases of non-menstrual toxic shock syndrome that can accompany surgical or injurious wound infections, as well as viral infections of the respiratory tract of influenza patients to which children are especially vulnerable [Schlievert (1993) ibid.; Tseng et al., Infect. Immun. 63:2880 (1995)]. Toxic shock syndrome, in its most severe form, causes shock and death [Murray et al., ASM News 61:229 (1995); Schlievert (1993) ibid.]. More generally, members of the staphylococcal exotoxin family, including SEA to SEE and toxic shock syndrome toxin 1 (TSST-1), have been implicated in toxic shock syndrome, in atopic dermatitis [Schlievert (1993) ibid.] and in Kawasaki's syndrome [Bohach et al., Crit. Rev. Microbiol. 17:251 (1990)].
Because of the potential for causing lethal shock in humans after aerosol exposure and because of the relative ease with which superantigen toxins can be produced in large amounts, there is concern that these toxins, alone or in combination, could be used as a biological weapon [Lowell et al., Infect. Immun. 64:1706 (1996)]. SEB is thought to be a potential biological weapon mainly in view of its lethal potential. However, through their exquisite ability to induce vomiting and diarrhea, staphylococcal and streptococcal superantigens are also incapacitating agents that could severely impair the effectiveness of a fighting force, even temporarily, thereby enhancing vulnerability to conventional military means. Moreover, mass incapacitation of civilians, accompanied by high morbidity if low mortality, constitutes a serious bio-terror threat. Needless to say, the harmful effects of SEB, and of other members of the superantigen exotoxin family, need to be generally attacked, and not only in connection with the military aspect.
Superantigens are toxic mitogens that trigger a paradoxical response in the infected organism: a vast stimulation of the immune system on the one hand and, on the other hand, a profound immunosuppression that may allow the multiplication of the infecting bacteria, unimpeded by an immune response [Hoffman, Science 248:685 (1990); Smith and Johnson J. Immunol. 115:575 (1975); Marrack et al., J. Exp. Med. 171:455 (1990); Pinto et al., Transplantation 25:320 (1978)]. During the cellular immune response, a dynamic interplay is induced, by antigens or mitogens, between activation of Th1 type cytokine gene expression, exemplified by interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β), and on the other hand, its cell-mediated suppression by CD8 cells and other cell subsets [Ketzinel et al., Scand. J. Immunol. 33:593 (1991); Arad et al., Cell Immunol 160:240 (1995)], and by the inhibitory cytokines from Th2 cells, IL-4 and IL-10 [Mosmann and Coffman, Annu. Rev. Immunol. 7:145 (1989)].
Bacterial superantigens are exotoxins that stimulate a 5- to 50-thousandfold greater proportion of rodent or human T cells than do ordinary antigens. Thus, SEB activates 30-40% of all T cells in some mice to divide and produce cytokines [Marrack and Kappler (1990) ibid.]. Indeed, toxicity of SEB requires T cells; mice that lack T cells or SEB-reactive T cells are not affected by doses of SEB that cause weight loss and death in normal animals [Marrack et al. (1990) ibid.; Marrack and Kappler (1990) ibid.]. Bypassing the restricted presentation of conventional antigens, superantigens produced by S. aureus and Streptococcus pyogenes bind directly to most major histocompatibility (MHC) class II molecules and activate virtually all T cells bearing particular domains in the variable portion of the T-cell receptor (TCR) β chain, without need for processing by antigen-presenting cells [Scholl, P. et al., Proc. Natl. Acad. Sci. USA 86:4210-4214 (1989); Fraser, J. D. Nature 339(6221):221-3 (1989); Choi, Y. W. et al., Nature 346(6283):471-3 (1990); Janeway, C. A. Jr. et al., Immunol. Rev. 107:61-88 (1989)]. This results in an excessive induction of T helper 1 (Th1) cytokines interleukin-2 (IL2), interferon-γ (IFN-γ) and tumor necrosis factor β, mediators of toxic shock [Marrack, P. and Kappler, J. Science 248:705-711 (1990a); Marrack, P. et al., J. Exp. Med. 171(2):455-64 (1990b); Miethke, T. et al., J. Exp. Med. 175(1):91-8 (1992); Hackett, S. P. and Stevens, D. L. J. Infect. Dis. 168:232-235 (1993); Arad, G. et al., Nat. Med. 6(4):414-21 (2000)]. Superantigens thus use the same ligands as conventional antigens but do so distinctly [Sundberg, E. J. et al., Structure (Camb) 10:687-699 (2002a); Sundberg, E. J. et al., Curr. Opin. Immunol. 14:36-44 (2002b)].
The toxicity of SEB and related exotoxins is thought to be related to the capacity of these molecules to stimulate a rapid and excessive production of cytokines, especially of IL-2, IFN-γ and tumor necrosis factors (TNFs). IL-2, IFN-γ, and TNF-β are secreted from activated T helper type 1 (Th1) cells while TNF-α is secreted by Th1 cells, monocytes and macrophages. High levels of these cytokines, suddenly produced, have been implicated as a central pathogenic factor in toxin-related toxicity [Schad et al., EMBO J. 14:3292 (1995)] and are thought to cause a rapid drop in blood pressure leading to toxic shock.
While investigation has produced a plausible explanation for the vast stimulation of T cells by SEs, it is not yet clear why these toxins are also strongly immunosuppressive. They induce a decline in both primary T and B cell responses, including the production of antibodies and the generation of plaque-forming cells [Hoffman (1990) ibid.; Smith and Johnson (1975) ibid.; Marrack (1990) ibid.; Pinto (1978) ibid.; Ikejima et al., J. Clin. Invest. 73:1312 (1984); Poindexter & Schlievert, J. Infect. Dis. 153:772 (1986)].
The sensitivity of humans to staphylococcal toxins exceeds that of mice by a factor of 100. Thus, the toxic shock syndrome toxin 1, TSST-1, another pyrogenic exotoxin from S. aureus, stimulates human T cells to express the key cytokines, IL-2, IFN-γ and TNF-β at <0.1 pg/ml, while murine cells require approximately 10 pg/ml [Uchiyama et al., J. Immunol. 143:3173 (1989)]. Mice may have developed relative resistance to toxic mitogens by deleting from their T cell repertoire those cells that display the most highly reactive V-β chains or by eliminating these V-β genes [Marrack and Kappler (1990) ibid.]. Such deletions have not been detected in humans, making them far more vulnerable.
The incapacitating and potentially lethal effects of SEB (and of exotoxins of the same family of superantigens) in humans, whether exerted on civilians or military personnel, create a need for prophylaxis against these toxins, and for treatment of toxin-exposed individuals.
Bacterial superantigens are among the most lethal of toxins, and they can be weaponized. These highly stable proteins resist boiling and are easy to produce and deliver. Bypassing the restricted presentation of conventional antigens, superantigens can activate up to 50% of T cells to divide and produce cytokines. Thus, superantigens activate the cellular immune response at least 5,000-fold more strongly than do ordinary antigens. Toxicity results from massive induction of Th1 cell-derived cytokines that include IL-2, IFN-γ and TNF. Death results within 24-48 hours, but even at concentrations several logs below lethal ones, these toxins severely incapacitate.
The family of superantigens produced by the common S. aureus and Streptococcus pyogenes (‘flesh-eating bacteria’) comprises well over 20 members, including staphylococcal enterotoxins SEA to SEE, among which SEB is most prominent, and toxic shock syndrome toxin 1 (TSST-1), and streptococcal pyrogenic exotoxins, inter alia SPEA. To compound the problem of protecting against superantigen-induced pathology, the amino acid sequences of superantigens are highly divergent: SEB and SEA have 28% homology, while TSST-1 exhibits only 6% sequence homology with SEB. The nature of toxins or toxin mixtures encountered during toxic shock, or in combat or bio-terrorism situations cannot be anticipated with certainty. The most likely scenarios of biological warfare entail not the use of a single, purified superantigen but rather of natural mixtures of superantigenic toxins, obtained by culturing the bacteria. This complexity demands the development of broad-spectrum countermeasures.
The inventors have previously explored the possibility of blocking superantigen action at the top of the toxicity cascade, before activation of T cells takes place. A purely intuitive approach has yielded the design of 12- or 14-amino acid peptide antagonists [p12A and p14A, also denoted by SEQ ID NO: 1 (daY N K K K A T V Q E L Dda) (“da” designates D-alaninedenoted by the ‘A’ in p 12A) and SEQ ID NO: 2 (daV Q Y N K K K A T V Q E L Dda), respectively] that inhibit induction of human Th1 cytokine gene expression by widely different superantigens (SEB, SEA, TSST-1 and SPEA), protect mice from the lethal effects of these toxins while allowing rapid development of broad-spectrum immunity against toxin challenge [Arad et al., Nature Medicine 6:414-421 (2000); Arad et al., J. Leuk. Biol. 69:921-927 (2001)], and protects pigs from incapacitation symptoms as are seen in humans in early toxic shock [applicant's Japanese Application No. 2001-377682 JP, and applicant's U.S. application Ser. No. 10/172,425]. Because pigs are closer to humans in their immune system than are mice and, unlike mice, require no presensitization to the toxic effect of superantigens, these findings support the expectation that with proper effort, efficacy in humans can be reached. No side effects of antagonist peptide were detected in mice or pigs. Antibodies against the antagonist could not be found; indeed, the small size and relatively rapid clearance of a short peptide (12-14 amino acids) constitute therapeutic advantages. The antagonist blocks the action of a superantigen (but not of a conventional antigen) on human lymphoid cells at a molar excess of about 100-fold, and prevents lethal shock in mice and incapacitation in pigs at a molar excess of only about 20- to 40-fold, implying that it binds tightly to its cellular target and that this target is critical for the superantigen-mediated activation of T cells. The antagonist activity of this peptide identified a novel superantigen domain that is critical for the superantigen action [Arad (2000) ibid.]. This finding raised the possibility that superantigens may use this domain to bind to a third receptor.
CD28 and B7-2 serve as principal costimulatory ligands for conventional antigens [reviewed by Lenschow, D. J. et al., Annu. Rev. Immunol. 14:233-58 (1996); Salomon, B. and Bluestone, J. A. Annu. Rev. Immunol. 19:225-52 (2001); Acuto, O. and Michel, F. Nat. Rev. Immunol. 3(12):939-51 (2003)]. The present invention now shows that to deliver the signal for Th1 activation, a superantigen must bind directly to CD28. Signaling is blocked by peptide mimetics of the contact region in each ligand: the β-strand-hinge-α-helix domain in superantigens [Arad (2000) ibid.; the ‘antagonist domain’] and two non-contiguous domains in CD28 that form the predicted homodimerization interface. Thus, due to the surprising direct interaction of CD28 and the superantigen, which was shown to be essential for Th1 lymphocytes activation, CD28 became the first drug target for treatment of lethal toxic shock. Interaction of an antagonist agent with this receptor allows it to block the action of superantigen toxins. Insight into the nature of this receptor target and of its interaction with the antagonist or with superantigen now provides a novel approach to design yet more effective antagonists.
Full activation of T cells is not solely dependent on the interaction of MHC class II molecule, superantigen and TCR. Sustained TCR engagement, although essential for T cell activation, faces many barriers. First, the TCR has a low affinity for antigens. Second, the number of antigenic complexes between the antigen-presenting cell and T cell can be very low.
Third, the movement of T cells works against sustained recognition of antigen. Although superantigens are far superior to ordinary antigens in overcoming these limitations and bypass MHC restrictions while binding to many TCR Vβ chains, they still require costimulatory ligands for T cell activation, including those of the B7 family on the antigen-presenting cell and CD28 and CTLA-4 on T cells.
While several investigators have suggested a role for B7-1 and B7-2 in the activation of T cells by a superantigen, the results were contradictory. For example, Muraille et al [Int. Immunol. 7:295-304 (1995)] reported that costimulation, by use of CD28- or B7-1-transfected cells, lowered the threshold for activation of naive T cells by bacterial superantigens. On the other hand, Muraille et al., [Cell. Immunol. 162:315-320 (1995a)] claimed that a combination of monoclonal antibodies to murine B7-1 and B7-2 molecules inhibits the in vitro response of naive T cells to SEA, SEB, and TSST-1. The inhibition of T cell responses required simultaneous blocking of B7-1 and B7-2, and they suggested that either B7-1 or B7-2 is sufficient to provide costimulatory signals to naive T cells in response to bacterial exotoxins. Inhibition of T cell activation by antibodies to B7-related molecules could be overcome by antibodies to CD28, raising the hypothesis that CD28-mediated signals participate in T cell activation by bacterial superantigens [Muraille (1995a) ibid.]. Yet another study by the same group, however, concluded that a single dose of anti-B7-2 antibodies, but not of anti-B7-1 antibodies, significantly inhibited T cell activation, and reduced the lethal effect of SEB in D-galactosamine-sensitized mice [Muraille et al., Eur. J. Immunol. 25:2111-2114 (1995b)]. Indeed, CTLA-4Ig or anti-B7-1 antibodies had little or no effect on superantigen-mediated activation of naïve T cells [reviewed by Muraille (1995b) ibid.]. These conclusions were subsequently rendered doubtful by a report, again from the same group, that blocking of CD80- or CD86-derived signals by specific monoclonal antibodies led to slower kinetics of IL-2 production in response to SEB [Muraille et al., Immunology 89:245-249 (1996)]. Krummel et al., [Int. Immunol. 8:519-523 (1996)] reported likewise that antibodies against B7-1/B7-2 or Fab fragments of anti-CD28 antibodies significantly inhibit the response of splenocytes to SEB. Mittrucker. et al., [J. Exp. Med. 183:2481-2488 (1996)] showed induction of unresponsiveness and impaired T cell expansion by SEB in CD28-deficient mice. The lack of expansion was not due to a failure of SEB to activate Vβ8+ T cells, as Vβ8+ T cells from both CD28−/− and CD28+/+ mice showed similar phenotypic changes within the first 24 h after SEB injection and cell cycle analysis showed that an equal percentage of Vβ8+ T cells started to proliferate. However, the phenotype and the state of proliferation of Vβ8+ T cells was different at later time points. They concluded that CD28 costimulation is crucial for the T cell-mediated toxicity of SEB. Protection against lethal toxic shock by targeted disruption of the CD28 gene was shown by Saha et al. [J. Exp. Med. 183:2675-2680 (1996)] who reported that CD28-deficient mice (CD28−/−) were completely resistant to TSST-1-induced lethal TSS while CD28+/− littermate mice were partially resistant to TSST-1. The mechanism for the resistance of the CD28−/− mice was a complete abrogation of TNF-alpha accumulation in the serum and a nearly complete (90%) impairment of IFN-gamma secretion in response to TSST-1 injection. In contrast, the serum level of IL-2 was only moderately influenced by the variation of CD28 expression. The hierarchy of TSST-1 resistance among CD28 wild-type (CD28+/+), CD28 heterozygous (CD28+/−), and CD28−/− mice suggested a gene-dose effect, implying that the levels of T cell surface CD28 expression critically regulate superantigen-mediated costimulation. Although these results demonstrated a primary and non-redundant role of CD28 receptors in the initiation of the in vivo cytokine cascade, and suggested therapeutic approaches for superantigen-mediated immunopathology, no concrete approach was suggested. Wang et al. [J. Immunol. 158:2856-2861 (1997)] claimed that CD28 ligation prevents bacterial toxin-induced septic shock in mice by inducing IL-10 expression. They observed that septic shock syndrome and death mediated by SEB could be prevented by administration of anti-CD28 antibodies. Anti-CD28 antibody treatment, they claimed, stimulated the expression of IL-10, both in splenocytes and in T cell lines. Furthermore, injection of anti-IL-10 could abolish the protective effect of anti-CD28 on septic shock. In the light of the findings presented herein, the results of Wang et al. (op. cit.) can be accounted for as follows: anti-CD28 inhibited SEB action in their experiments not by inducing IL-10, as they claimed, but by blocking the direct binding of SEB to CD28 which, as the inventors have now shown, is obligatory for the induction of Th1 cytokine gene expression by SEB that in turn results in lethal shock, without interfering with the induction of the Th2 cytokine IL-10 by the superantigen which, as the inventors have shown, is independent of CD28 engagement. Thus, the action of SEB was apparent to Wang et al. (ibid.) only in elicitation of a Th2 response that was protective. Wang et al. neither showed nor suggested direct binding of SEB to CD28 and they neither showed nor suggested that such binding is needed selectively for a Th1 response but not for a Th2 response. Indeed, Wang et al. teach away from direct binding of SEB to CD28 and from the concept that such binding is needed selectively for a Th1 response but not for a Th2 response, as shown by the inventors. This is seen, for instance, from the title of Wang et al. “CD28 ligation prevents bacterial toxin-induced septic shock in mice by inducing IL-10 expression” and from their sentence “anti-CD28 Ab treatment stimulated the expression of IL-10, both in splenocytes and in T cell lines”. In view of the prior art and Wang et al. [ibid.], the novel results obtained by the inventors are surprising. The present results do not bear out the claims of Wang et al. (ibid.). Indeed, as shown below in FIG. 10, anti-CD28 mAb (monoclonal antibody) fails to induce expression of IL-10 in human peripheral blood mononuclear cell (PBMC) populations. Moreover, antagonist peptide leaves the induction of IL-10 by superantigen intact (FIG. 3).
In summary, the prior art indicates that:    1. B7-1 and B7-2 engage T cells but the role of each coligand in terms of activating Th1 or Th2 cells has remained controversial;    2. The role of B7-1 and B7-2 in T cell activation by superantigen toxins has also remained controversial, with some reports claiming that either B7 ligand will costimulate superantigen action while other reports advocate a role for B7-2;    3. CD28 acts as a costimulatory ligand for superantigens, as it does for conventional antigens that are presented by the MHC class II molecule;    4. The mechanism of costimulation by CD28, in conjunction with B7-1 and/or B7-2, of superantigen-mediated T cell responses is not known but is thought to be similar for superantigens and conventional antigens, and more specifically, the prior art teaches away from the concept that the mechanism of costimulation of superantigens and of conventional antigens could be different;    5. The specific roles of B7-1 and B7-2, respectively, in the CD28-mediated activation of Th1 and Th2 responses by superantigens are unknown.
SEB binds to the MHC class II α chain with low affinity (Kd, 0.34 μM) [Papageorgiou, A. C. et al., EMBO J. 18:9-21(1999)]. Superantigens bind even more weakly to the TCR, with affinities in the 1-100 μM range [Leder, L. et al., J. Exp. Med. 187:823-33 (1998); Andersen, P. S. et al., Biol. Chem. 276: 33452-7 (2001); Redpath, S. et al., J. Immunol. 163: 6-10 (1999)].
A low affinity and high off-rate, which determines the average time of ligand/receptor contact, is thought to determine the signaling strength through the TCR. Indeed, mutant forms of the staphylococcal superantigen SEC3 having increased affinity for TCR Vbeta8.2 domains also showed increased mitogenic potency on T cells [Andersen (2001) ibid.]. A direct correlation was found between the binding affinity of SEC3 variants for the TCR and the strength of the T cell response they evoke. This finding would suggest that superantigens could have evolved higher affinities for the TCR. That has not occurred in nature; instead, even a potent superantigen such as SEA retains a low affinity for the TCR [Kieke, M. C. et al., J. Mol. Biol. 307:1305-15 (2001)]. Surface plasmon resonance studies show that in absolute terms, the interaction of superantigens with either individual ligand, MHC class II molecule or TCR, is very weak [Redpath. (1999) ibid.; Seth, A. et al., Nature 369: 324-7 (1994)].
This suggests that to achieve T cell activation, superantigens may need to rely on additional ligand interactions, and the antagonist peptides described in the present application, block the interaction of superantigen with the CD28 receptor, an interaction that is critical for superantigen-mediated activation of the harmful Th1 cytokine response.
Occupation of the TCR binding domain on superantigens would be another strategy to block the action of superantigens. Using this approach, a soluble mutant form of the TCR Vbeta8 chain was selected that binds SEC3 1000-fold more tightly (Kd of 7 nM) [Kieke (2001) ibid.,]. This mutant Vbeta8 protein antagonized SEC3-mediated specific T cell activity. Unlike the short antagonist peptide described herein, the soluble Vbeta8 protein is of high molecular weight and thus will be more difficult to deliver. Even more problematic is the fact that different superantigens bind to the TCR with molecular surfaces that can differ widely. Moreover, each superantigen binds preferentially only to a narrow, individual subset of the Vbeta chain repertoire [Kieke (2001) ibid.,; Kline, J. B. et al., Mol. Microbiol. 24:191-202 (1997); Li, H. et al., Immunol. Rev. 163:177-86 (1998)].
Indeed, for different superantigens, highly efficient T cell activation may be achieved through structurally diverse strategies of TCR ligation [Sundberg, E. J. et al., Structure (Camb) 10:687-99 (2002)]. Hence, a soluble mutant Vbeta8 protein may exhibit limited specificity for superantigens.
As indicated above, CD28 belongs to a triad of costimulatory ligands whose genes are tightly linked: CD28, cytotoxic T-lymphocyte-associated protein 4 (CTLA4)(CD152) and inducible costimulator (ICOS) [reviewed by Sharpe, A. H. and Freeman, G. J., Nat. Rev. Immunol. 2(2):116-26 (2002); Carreno, B. M. and Collins, M. Annu. Rev. Immunol. 20:29-53 (2002)]. Via their coligands from the B7 family, these proteins function as costimulatory receptors that regulate signaling by ordinary antigens. CD28 acts as the critical early signal transducer for the innate immune response, balanced by ICOS and CTLA4 [reviewed by Rudd, C. E. and Schneider H. Nat. Rev. Immunol. 3(7):544-56 (2003)]. The present invention now further shows that through its β-strand-hinge-α-helix domain, the major superantigen staphylococcal enterotoxin B (SEB) binds with high affinity to each member of this conserved receptor family. Peptides derived from either rim of the bipartite dimer interface in CTLA4 [Schwartz, J. C. et al., Nature 410:604-608 (2001); Stamper, C. C. et al., Nature 410(6828):608-11 (2001)] or in CD28 and ICOS as predicted by sequence alignment, although unique for each costimulatory receptor, are potent antagonists that block superantigen-mediated induction of human Th1 cytokine gene expression and protect mice from lethal challenge with SEB. Apparently, the mode of action of these peptides is to compete with CD28 for its binding site in superantigens. SEB induces a vigorous expression of Th1 and Th2 cytokine genes but only induction of the Th1 response is dependent on CD28 signaling.
Direct binding to CD28 underlies the toxicity of the superantigens. The findings of the present invention reveal a mechanism of subversion of the innate immune response in which the superantigen co-opts a costimulatory ligand of the host for use as its obligatory receptor. This strategy may be used more widely by pathogens.
Therefore, it is an object of the invention to provide methods for inhibiting the activation of a T cell costimulatory pathway, preferably, the CD28/B7 pathway by a pathogenic agent, in a subject in need thereof. Such methods are based on the use of a substance which inhibits the direct interaction of a component derived from said pathogenic agent and a binding site within a T cell costimulatory pathway member molecule, which site is derived from the dimer interface of said T cell costimulatory pathway member.
Another object of the invention is to provide substances, preferably peptides, which inhibit the direct interaction of a component derived from the said pathogenic agent and a binding site within the dimer interface of a T cell costimulatory pathway member, preferably, CD28, CTLA4 and ICOS. Such peptides are provided by the invention and include peptides comprising an amino acid sequence derived from a dimer interface of a T cell costimulatory pathway member, for example the peptides of SEQ ID NO: 5, 15, 16, 18, 19, 20, 21, 59 and 60, and also peptides comprising an amino acid sequence which specifically binds to an amino acid sequence within the dimer interface of a T cell costimulatory pathway member, for example the peptides of SEQ ID NO: 12, 13, 14 and 27 to 58.
Another object of the invention is to provide compositions and method of treatment of immune-related disorders caused by a pathogenic agent, particularly, a superantigen exotoxin.
It is yet a further object of the present invention to use CD28 as a powerful novel target for the development of antidotes to superantigen-induced toxic shock symptoms, whether septic shock, toxic shock or incapacitation by toxin.
These, and other objects of the invention will become apparent as the description proceeds.